NMR and HPLC-NMR spectroscopic studies of futile deacetylation in paracetamol metabolites in rat and man

Abstract

HPLC-NMR spectroscopy has been used to investigate the level of deacetylation followed by reacetylation (futile deacetylation) of metabolites of paracetamol detected in human and rat urine. This has been achieved through the synthesis and administration of paracetamol isotopically labeled at the acetyl group with C 2H 3, 13CH 3 and 13CO- 13CH 3. Using paracetamol-C 2H 3 it had been shown that in the rat the sulphate metabolite present in the urine shows 10–13% futile deacetylation depending on the dose, whereas for paracetamol- 13CO- 13CH 3 the corresponding value was about 8%. After solid phase extraction, it was also possible to determine the level of futile deacetylation in the glucuronide metabolite using directly-coupled HPLC-NMR. This approach was facilitated by the use of acetonitrile-d 3 as an HPLC eluent and the HPLC-NMR analyses showed that the level of futile deacetylation in the sulphate and glucuronide metabolites were equal at about 9%. The glucuronide of paracetamol-C 2H 3 was the predominant metabolite in man and following separation using HPLC-NMR, the level of futile deacetylation was shown to be 1% for the glucuronide and 2% for the sulphate, these values being equal within experimental error. This work demonstrates the utility of NMR and HPLC-NMR spectroscopy for isotope exchange studies.